Polypeptide possessing thymic activity

ABSTRACT

The novel thymic factor polypeptide hormone, elaborated by the thymus and und in the blood serum, is isolated. The hormone is useful for the treatment of autoimmune diseases and for selectively stimulating T-cell activity in aging subjects.

This application is a continuation-in-part of co-pending applicationU.S. Ser. No. 454,493, filed Mar. 25, 1974, now abandoned.

BACKGROUND OF THE INVENTION

It has been reported that the injection of thymic cell-free extracts canrestore the immunological function of neonatally thymectomized mice toreject skin grafts. Data showing partial reconstitution of neonatallythymectomized mice by thymus grafts in a Millipore chamber has suggestedthat the thymus acts as an endocrine gland and elaborates a hormone intothe blood circulation. [See D. Osoba, et al., Nature 199, 653 (1963)].The present invention is directed to this novel polypeptide hormone,designated thymic factor polypeptide hormone, and a novel method for theisolation and purification of this medicinally useful polypeptide.

SUMMARY OF THE INVENTION

In general, the hormone is obtained from pig blood by defibrination,dialysis and concentration on a suitable filter followed byfractionation through a molecular sieve, chromatography on an ionexchange resin, further fractionation by thin layer chromatography andfinally by electrophoresis. The thin layer chromatography andelectrophoresis can be substituted by further gel filtration steps. Eachstep of the isolation is monitored by a bioassay which is based on theproperty of the peptide which inhibits the formation of rosettes in thepresence of azathioprine (hereinafter designated AZ). The process isuseful for isolating thymic factor polypeptide hormone from human, mouseand bovine as well as porcine blood. The preferred source in the presentinvention is porcine blood.

According to the process of the present invention freshly collectedporcine blood is defibrinated by mechanical means. This can beaccomplished by agitating the freshly collected blood with a stirringrod and removing the fibrin adhering to the rod.

The defibrinated blood is dialyzed to remove the bulk of the protein andother non-dializable material. This is conveniently accomplished by theuse of an artifical kidney (hereinafter referred to as a dialyzer)equipped with a suitable membrane such as a polyacrylamide membrane. Adialyzer is particularly suitable for dialyzing large volumes. Thedefibrinated blood is centrifuged to obtain serum prior to dialysis.

The dialysate, which contains the thymic factor, is concentrated on anultrafiltration membrane. Ultrafiltration (or diafiltration) refers tothe selective retention of solutes by convective solvent flow through ananisotropic "skinned" membrane. In ultrafiltration, solutes, colloids orparticles of dimensions larger than the specified membrane "cut-off" arequantitatively retained in solution, while solutes smaller than theuniform minute skin pores pass unhindered with solvent through thesupportive membrane substructure. Thymic factor polypeptide hormone ispreferably concentrated by an Amicon UM2 ultrafiltration membrane.

Amicon UM2 ultrafiltration membranes are unique structures cast in flatsheet form from a non-cellulosic polymer solution. These membranes areanisotropic consisting of a very thin (0.1 to 1.5 μm), dense "skin" ofextremely fine, controlled pore texture on a much thicker (50 to 250μm), open-celled spongy layer of the same polymer. The UM2 membrane hasa "cut-off" of molecular weight about 1,000, i.e., it retains substanceshaving a molecular weight greater than about 1,000 and allows passage ofmolecules less than about 1,000 molecular weight. This results in theconcentration of the thymic factor polypeptide hormone in thediafiltration chamber. Amicon membranes are manufactured by AmiconCorporation, 21 Hartwell Avenue, Lexington, Massachusetts.

The concentrated solution containing thymic factor is subjected to gelfiltration through a dextran gel molecular sieve. A suitable dextran gelis Sephadex gel manufactured by Pharmacia Fine Chemicals, Box 175, S-75104, Uppsala 1, Sweden. Sephadex is a chromatographic material capable ofseparating substances according to molecular size. The separation methodis most commonly known as gel filtration or gel chromatography. Sephadexis a bead-formed, dextran gel prepared by cross-linking selected dextranfractions with epichlorohydrin. Dextran is an anhydroglucose polymerproduced in sucrose-containing solutions by different strains ofLeuconostoc mesenteroides. Because of the high content of hydroxylgroups in the polysaccharide chains, Sephadex is strongly hydrophilicand thus swells in water and electrolyte solutions.

Various types of Sephadex are available, differing in their swellingproperties. Each Sephadex type fractionates within a particularmolecular weight range, determined by the degree of swelling of the gel.Molecules of a molecular weight above the upper limit of this range -the exclusion limit - are totally excluded from the gel and are elutedat the void volume. Molecules of a molecular weight below thefractionation range are usually eluted at an elution volumeapproximately equal to the total bed volume. Sephadex G-25, thepreferred Sephadex for fractionating thymic factor, has thefractionation range of molecular weight 1,000 to 5,000. The "fine" gradeis preferred for preparative purposes, where the extremely goodresolution that can be achieved with the "superfine" grade is notrequired, but where the flow rate is of greater importance.

The Sephadex G-25 is eluted with a suitable phosphate buffer. Residualproteins still present in the preparation are not retarded in thiscolumn and are eluted at the void volume.

The active fractions, as determined by activity in the rosette assay,are pooled, desalted and subjected to chromatography on an ion exchangecellulose preferably a cation exchange resin such as carboxymethylcellulose (CM-cellulose). Such a suitable carboxymethyl cellulose isobtained from Brown Company, 500 Fifth Avenue, New York, New York. Thecarboxymethyl cellulose column is eluted with a salt gradient. Theactive fractions may optionally be filtered through a Millipore filterto remove bacteria in which case a suitable preservative agent, such assodium azide, is added to all subsequent solutions to maintainsterility.

Any number of cellulose or derivatives of cellulose such asnitrocellulose discs with uniform porosity in the range of 0.03 to 3microns such as those provided by Millipore Corp., 200 Walsh Road,Bedford, Massachusetts, can be used for the microbial filtration.

The active fractions, determined by the rosette assay, are subjected toone dimensional and two dimensional thin layer chromatography and thenelectrophoresis. In the preferred process of the present invention, theactive fractions from the carboxymethyl cellulose column are subjectedto gel filtration using Sephadex G-25 eluted with dilute aqueous aceticacid and the active fractions from this column gel filtered throughSephadex G-10. Sephadex G-10 excludes molecules larger than molecularweight 700, accordingly thymic factor polypeptide is eluted at the voidvolume.

The novel polypeptide obtained by the above process is useful in thetreatment of autoimmune diseases such as lupus like pathology andspecifically for the treatment of Lupus Erythematosus in man. This novelpolypeptide is also useful for selectively stimulating T-cell activityin aging subjects.

The detailed description for the methods of isolating and purifying thepolypeptide hormone of this invention from porcine blood is set forth inExamples 1 and 2. The process is useful in isolating the thymic factorpolypeptide hormone from human, mouse and bovine as well as porcineblood. The polypeptide hormone is absent in the serum of thymectomizedpigs one week after thymectomy and it is present in serum of pigs thathave been sham thymectomized. The hormone is species non-specific andhence hormone isolated from porcin, mouse and bovine blood is equallyuseful in the treatment of humans.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Isolation andPurification of the Thymic Factor Polypeptide

Isolation of the thymic factor polypeptide is achieved by the successiveten operations:

(1) defibrination

(2) dialysis

(3) concentration on Amicon UM2 membrane

(4) Sephadex G-25 gel filtration

(5) chromatography on carboxymethyl cellulose (CM-cellulose)

(6) treatment with activated charcoal

(7) thin layer chromatography (TLC) in butanol; pyridine solvent

(8) two dimensional TLC: first dimension in 0.01N acetic acid and seconddimension in methanol; chloroform; ammonium hydroxide solvent

(9) rechromatography in methanol; chloroform; ammonium hydroxide solventand

(10) electrophoresis.

Each of these steps is described in detail below. The fractionationprocedure employs a starting batch of 8 liters of pig blood.

STEP (1) - SERUM PREPARATION BY DEFIBRINATION AND CENTRIFUGATION

Normal 3-4 month old pigs were bled lethally in a local abattoir and theblood was immediately defibrinated by mechanical agitation. The bloodwas cooled to 4° C. and transported to the laboratory where it wascentrifuged to obtain the serum.

Eight liters of defibrinated blood, centrifuged once for 15 min. at 4°C. at 1700x gravity, yielded 3.6 liters of serum.

Biological activity: The biological activity of defibrinated andcentrifuged serum as determined by the rosette assay (described below)was 1/128.

STEP (2) - DIALYSIS

3.6 liters of serum were dialysed in the cold room (4° C.) underultrafiltration conditions (positive pressure in the membrane, no liquidin the outside of the membrane) for four hours. The membrane used was apolyacrylonitrile membrane (a product of Rhone-Poulenc) with apermeability of 0.36 to 0.45 ml./min./mm² /mm. Hg pressure, with a totalsurface area of 1.02 square meters. The membrane was employed in acommercially available Rhone-Poulenc 6 dialyzer. (Rhone-Poulenc, Centrede Recherche Nicolas Grillet, Vitry sur Seine, France.) The serum wascirculated with a pump and the ultrafiltrate was collected in theoutside compartment of the dialyzer by a second pump equipped with anautomatic outflow and pressure device. 0.5% of protein was found in theultrafiltrate (80 mg./ml. in initial serum and 0.4 mg./ml. in theultrafiltrate). 2.8 l. of ultrafiltrate was obtained from 3.6 l. ofserum. Biological activity: The activity of the ultrafiltrate was 1/128in the rosette assay.

STEP (3) - AMICON UM2 MEMBRANE DIAFILTRATION

Four hundred ml. of serum ultrafiltrate was placed in each of sevenAmicon diafiltration chambers (model 402) and a continuous pressure of50 psi applied. After 8 hours, when the membrane was dry the chamber wasfilled with 4 ml. of phosphate buffer (0.2M, pH 7.3) and agitated 1minute to take up the polypeptide into the buffer.

Biological activity: The activity, as determined by the rosette assay,was enriched to 1/25,000. The 4 ml. washings were pooled and subjectedto Sephadex G-25 gel filtration.

STEP (4) - SEPHADEX G-25 GEL FILTRATION

The pooled sample of 28 ml. was applied on a 100 cm. × 5 cm. columnpacked with "fine" Sephadex G-25 and eluted with phosphate buffer (0.2M,pH 7.3). The flow rate was maintained at 5 ml./min., and 5 ml. fractionswere collected. The void volume (Vo) assessed by using Dextran Blue was580 ml. The bulk of protein contained in the sample was removed withelution volumes ranging from 540 ml. to 580 ml.

Biological activity: Fractions active in the rosette assay were found atan elution volume (Ve) of 1250 ml., with a Ve/Vo of 2.1. Activefractions included 15 ml. active at 1/256,000 and 30 ml. active at1/128,000.

STEP (5) - CARBOXYMETHYL CELLULOSE (CM) CHROMATOGRAPHY

The active fractions obtained in the Sephadex chromatography weredesalted using Amicon UM2 membranes as previously described by applyingthese fractions (45 ml. in total) together with 300 ml. of distilledwater to the Amicon unit until the liquid had completely passed throughthe membrane. The filter retained material was taken up in 7 ml. ofphosphate buffer (0.01M, pH 6.3). This sample was applied on a 15 × 0.9cm. column packed with CM-52 cellulose (Whatman). The cellulose was usedin its precycled form, and equilibrated with phosphate buffer (0.02M, pH6.3). After adding 40 ml. of 0.01M, pH 6.3 phosphate buffer to removeunbound material and to reequilibrate the column, the column was elutedwith a stepwise NaCl gradient, from 0.01M to 0.4M.

Biological activity: Less than 5% of biological activity was detected inthe volume eluted before NaCl was added. All the rest of the biologicalactivity was eluted as a single peak with NaCl molarity at 0.12M. Oneml. fractions were collected. The active fractions included twofractions of 1 ml. each active at 1/256,000; four fractions active at1/512,000 and a fraction active at 1/256,000. These fractions werepooled and treated with activated charcoal.

STEP (6) - TREATMENT WITH ACTIVATED CHARCOAL

In order to remove products susceptible to bind to activated charcoal,the CM cellulose eluted sample was incubated at 4° C. for 15 min. withactivated charcoal (1 ml. of activated charcoal at 50 mg./ml. water with1 vol. of CM cellulose active fractions). The activated charcoal wasremoved by centrifugation. The supernatant showed no change inbiological activity.

STEP (7) - PREPARATIVE THIN LAYER CHROMATOGRAPHY

The activated charcoal treated material was desalted on Amicon UM2membrane as previously described and the filter retained material wastaken up in water and lyophilized. The lyophilizate was taken up in 50μl. of water and applied in a horizontal line on a preparative cellulosechromatography plate. After development in butanol-pyridine (60:30) anarrow strip on the edge of the plate was visualized with fluorescamineor ninhydrin and the areas of the plate corresponding to thefluorescamine spots were eluted.

Fluram, a Hoffman La Roche tradename for fluorescamine, was sprayed asan acetonic solution at 15 mg.% and the spots were visible whenilluminated with a U.V. lamp. Spots were marked soon after sprayingsince it was observed that they do not persist longer than 1 to 2 min.Peptide staining was also obtained by spraying with a solution ofninhydrin-cadmium acetate. The cellulose strip of 1 cm. in widthcorresponding to the location of the fluorescamine spot was removed fromthe unstained portion of the plate and eluted. Elution was performed bymechanical agitation at 4° C. (1 cm.² of cellulose per 5 ml. H₂ O) for18 hours. The sample was tested for biological activity in the rosetteassay. 30 ml. were active at 1/25,000.

After developing with butanol-pyridine (60:30) solvent, no more than 2%of the initial activity was found at the origin and an activity notdistinguishable from 100% was found at the solvent front. The strip ofcellulose at the solvent front was eluted and the eluate lyophilized.

STEP (8) - TWO DIMENSIONAL PREPARATIVE THIN LAYER CHROMATOGRAPHY

A second TLC purification step was effected using two dimensionalchromatography on cellulose plates. The first solvent system was 0.01Nacetic acid in which the biological activity was found at R_(f) 0.8 andthe second solvent system was methanol, chloroform, ammonium hydroxide(20:20:9). Three spots were still observed with fluorescamine afterdevelopment in the second dimension. When eluted, the spot at R_(f) 0.32showed all the biological activity. 30 ml. of eluate was active at1/20,000.

STEP (9) - RECHROMATOGRAPHY

The eluted material showed only one spot when rechromatographed inmethanol; chloroform; ammonium hydroxide (20:20:9) and showed only onespot with fluorescamine or ninhydrin. When eluted, 30 ml. was active at1/20,000.

STEP (10) - HIGH VOLTAGE ELECTROPHORESIS

High voltage electrophoresis was used as the last step of purification.The paper electrophoresis was performed in formic acid diluted to pH1.9, at 40 Volts/cm. and 40 mA, for 50 minutes. Three components werevisualized by ninhydrin and fluorescamine. Under these conditions theactive product showed a cathodic migration of 9 cm. The active samplewas eluted from the paper with distilled water (1 ml. per 1 cm.² ofpaper). 4 ml. of eluate was active at 1/128,000 and 30 μg. of thymicfactor polypeptide hormone was recovered when assayed by ninhydrin andfluorescamine evaluated after hydrolysis overnight (in 6N HCl at 110°C.).

EXAMPLE 2 Isolation and Purification of the Thymic Factor Polypeptide

Isolation of the thymic factor polypeptide is also achieved by thefollowing seven operations:

(1) defibrination

(2) dialysis

(3) concentration on Amicon UM2 membrane

(4) Sephadex G-25 gel filtration and filtration through a Milliporemembrane

(5) chromatography on carboxymethyl cellulose (CM)

(6) sephadex G-25 gel filtration

(7) Sephadex G-10 gel filtration.

Each of these steps is described below in detail. The fractionationprocedure employed a starting batch of 2000 liters of porcine blood.

STEP (1) - DEFIBRINATION OF PIG BLOOD

Normal 3-4 month old pigs were bled lethally in a local abattoir and theblood was immediately defibrinated by mechanical agitation. The bloodwas cooled to 4° C. and transported to the laboratory where it wascentrifuged to obtain the serum. Eight liters of defibrinated blood,centrifuged once for 15 minutes at 4° C. at 1700x gravity, yielded 3.6liters of serum.

STEP (2) - SERUM PREPARATION BY ULTRAFILTRATION

Defibrinated pig blood (2000 liters) was subjected to ultrafiltrationusing a dialyzer under the conditions set forth in Example 1, Step 2.

STEP (3) - AMICON UM2 MEMBRANE DIAFILTRATION

Four hundred twenty liters of the serum ultrafiltrate was concentratedto a volume of 4.2 liters by diafiltration on Amicon UM2 membranes underthe conditions set forth in Example 1, Step 3). This represents a 100xincrease in concentration.

STEP (4) - SEPHADEX G-25 FILTRATION

A 3.0 liter portion of the Amicon concentrate was subjected to gelfiltration in 28 ml. portions on 107 Sephadex G-25 columns under theconditions set forth in Example 1, Step 4). The active fractions weredetected by the rosette assay and filtered through a Millipore membrane(pore size 0.22 microns) to remove contaminating bacteria. Sodium azidewas added to the extent of 0.02% to these active fractions and in theremaining steps below to maintain a sterile preparation.

STEP (5) - CARBOXYMETHYL CELLULOSE (CM) CHROMATOGRAPHY

The active fractions obtained by Sephadex G-25 chromatography, as setforth in Step 4 above, were desalted and chromatographed oncarboxymethyl cellulose as set forth in Example 1, Step 5).

The active fractions from each carboxymethyl cellulose column weredetected by the rosette assay and pooled. The pooled fractions from eachcarboxymethyl cellulose column were lyophilized.

STEP (6) - SEPHADEX G-25 GEL FILTRATION

The lyophilized fractions, obtained in Step 5) above, were subjected togel filtration on 100 columns of Sephadex G-25 packed in 5% acetic acidand having dimensions 90 cm. × 1.5 cm. and a void volume (Vo) of 44 ml.assessed by using Dextran Blue. A flow rate of 0.2 ml./min. wasmaintained and 2 ml. fractions were collected.

Biological activity: - Fractions active in the rosette assay were foundto be centered at an elution volume of 70 ml. (Ve) with a Ve/Vo of 1.6.The active fractions included 14 ml. active at 1/4 × 10⁷ and 4 ml. at1/1 × 10⁷. The active fractions were pooled, lyophilized withoutdesalting and subjected to gel filtration on Sephadex G-10.

STEP (7) - SEPHADEX G-10 GEL FILTRATION

The active fractions were subjected to gel filtration on twenty SephadexG-10 columns packed in distilled water and eluted with distilled water.The thymic factor was eluted at the void volume. The fractionscontaining active material were pooled and lyophilized to yield a totalof 100 nmoles of thymic factor peptide.

DETERMINATION OF SERUM THYMIC ACTIVITY BY THE ROSETTE ASSAY

The rosette assay has been previously described in J. F. Bach and M.Dardenne, Immunology, 25, 353 (1973), the contents of said article isbeing herein incorporated by reference.

Thymic activity is determined in serum by first filtering the serumthrough an Amicon membrane (Centriflo CF50 A, Amicon) through whichmolecules with a molecular weight lower than 50,000 can pass. Thefiltered serum is incubated in a haemolysis tube with 3 × 10⁶ spleencells obtained from adult C 57/B1 6 mice (obtained from Centre d'Elevagedes Animaux de Laboratoire du C.N.R.S. (45 Orleans, La Source))thymectomized 10 to 20 days before. The method of thymectomy isdescribed in M. Dardenne and J. F. Bach, Immunology, 25, 343 (1973) onpage 344. The contents of said article is being herein incorporated byreference.

The incubation is carried out for 90 minutes at 37° C. in the presenceof azathioprine (AZ) at a concentration of 10 μg./ml. Such aconcentration is intermediate between the AZ minimal concentrationinhibiting 50 percent spleen rosette forming cells (RFC) from normalmice (1 μg./ml.) and from adult thymectomized mice (25-10 μg./ml.). Atthe end of the incubation, 12 × 10⁶ sheep red blood cells (SRBC) areadded to the cells in the test sample. The cells in the sample arecentrifuged for 6 minutes at 200 g. and carefully and gently resuspendedby rotation on a roller (10 cm. diameter) at low speed (10 rev./min.).RFC are counted in a haematocytometer. In the absence of thymicactivity, the number of RFC is 1210/10⁶ cells ± 120 (standard deviation,SD). In the presence of thymic activity, the RFC level is lowered to 200to 400/10⁶ cells. In the absence of AZ normal serum induced noinhibition of RFC. Thymic activity is defined as inhibition of more than50% rosette forming cells.

PROPERTIES OF POLYPEPTIDE HORMONE

The polypeptide hormone of this invention has an isoelectric point (pI)at pH 7.5 ± 0.1. Lack of affinity to activated charcoal indicates theabsence of aromatic amino acids. Elution of the polypeptide with thevoid volume when chromatographed on Sephadex G-10 and retention onSephadex G-15, G-25 and G-50 indicates a molecular weight greater than700 and less than 3,000. Ultrafiltration through Amicon membranesindicates a molecular weight of 500 to 10,000. No significant retentionof the polypeptide is observed on CF-50, PM-30 and PM-10 membranes whichhave molecular cut-offs of 50,000; 30,000 and 10,000 respectively.Conversely no significant activity is found in UM2 and UM0.5 filtrateswhich have molecular weight cut-offs of 1,000 and 500 respectively. Thepolypeptide has the property that it binds to carboxymethyl cellulose atpH lower than 7.0 and to DEAE cellulose at a pH higher than 9.0. Thepolypeptide bound on carboxymethyl cellulose can be eluted with 0.12MNaCl. Chromatography on cellulose plates shows an R_(f) 0.32 inmethanol; chloroform; ammonium hydroxide (20:20:9) solvent; R_(f) 1.0 indistilled water and R_(f) 0.8 in 0.01N acetic acid.

Acid hydrolysis at 110° C. in 6N HCl overnight yielded the followingamino acid composition: Asp,Ser₂,Glu₂,Lys,Gly₂,Ala wherein theabbreviations have the following definitions:

    ______________________________________                                        Abbreviation        Amino Acid                                                ______________________________________                                        Asp                 aspartic acid                                             Ser                 serine                                                    Glu                 glutamic acid                                             Lys                 lysine                                                    Gly                 glycine                                                   Ala                 alanine                                                   ______________________________________                                    

In view of the molecular weight of the order of 1,000 daltons, anisoelectric point pI at about pH 7.5, and the amino acid analysis andthe sequencing studies carried out using the Edman technique modified byHartley, it is believed that the amino acid sequence of thymic factor isas follows: pyroglutamyl-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn wherein theabbreviations have the following definitions:

    ______________________________________                                        Abbreviation        Amino Acid                                                ______________________________________                                        Gln                 glutamine                                                 Asn                 asparagine                                                ______________________________________                                    

The novel polypeptide of this invention may be administeredintravenously or intramuscularly. Suitable carriers which may be used inthe composition include, for example, sterile liquids such as water orsaline. Also, in addition to a carrier the instant compositions may alsoinclude other ingredients such as stabilizers, antioxidants, suspendingagents, or preservatives such as phenol or chlorobutanol and the like.The finished solution can be easily sterilized by conventionalfiltration techniques.

The composition used in this invention contains in aqueous solution asufficient quantity of the therapeutic agent to be medicinally useful.The dosage to be administered depends to a large extent upon thecondition of the subject being treated and the weight of the host. Theparenteral route being preferred. In general, the useful daily dosageconsists of from about 0.0001 to about 1.0 mg. of active ingredient perkg. of body weight of the subject in one or more applications per day. Apreferred daily dosage lies in the range of from about 0.0001 to 0.01mg. of active ingredient per kg. of body weight. An injectable dose ofparticular interest is 0.1 mg. of active material, administered daily.In parenteral administration the unit dosage is usually the purecompound in a sterile water solution or in the form of a soluble powderintended for solution.

The following example describes composition for parenteraladministration packaged in ampoules, vials and multiple dose vials.

EXAMPLE 3

    ______________________________________                                        Parenteral Solution Containing 5.0 mg. of Thymic                              Factor Polypeptide Hormone                                                    ______________________________________                                        Thymic Factor Polypeptide Hormone                                                                      0.1 mg.                                              Pyrogen free sterile distilled water                                                                   1.0 ml.                                              ______________________________________                                    

Sterilized by filtration and packaged in ampoules, vials, or multipledose vials.

EXAMPLE 4

    ______________________________________                                        Ampoules Containing 5.0 mg. of Lyophilized Thymic                             Factor Polypeptide Hormone                                                    ______________________________________                                        Ampoule:                                                                      Thymic Factor Polypeptide Hormone                                                                      0.1 mg.                                              Ampoule:                                                                      Diluent: Sterile Water for Injection                                                                    1 ml.                                               ______________________________________                                    

Appropriate multiples of the above amounts are used as required.

What is claimed is:
 1. A method of producing a polypeptide hormonehaving thymic activity which comprises:(a) dialyzing defibrinated bloodserum under ultrafiltration conditions through a polyacrylonitrilemembrane having a permeability of 0.36 to 0.45 ml./min./mm² /mm Hgpressure, (b) passing the dialyzate through an Amicon UM2 filtermembrane which allows passage of molecules of less than 1000 molecularweight, and collecting the material retained by the filter membrane, (c)chromatographing the filter-retained material through Sephadex G-25 andeluting with 0.2M phosphate buffer at pH 7.3, (d) desalting said eluateand chromatographing the desalted product in phosphate buffer at pH 6.3on carboxymethylcellulose and eluting with a NaCl gradient, (e)chromatographing said eluate through Sephadex G-25 and eluting with 5%acetic acid, (f) chromatographing said eluate through Sephadex G-10 andeluting with water.
 2. A method of producing a polypeptide hormonehaving thymic activity which comprises:(a) dialyzing defibrinated bloodserum under ultrafiltration conditions through a polyacrylonitrilemembrane having a permeability of 0.36 to 0.45 ml./min./mm² /mm Hgpressure, (b) passing the dialyzate through an Amicon UM2 filtermembrane which allows passage of molecules of less than 1000 molecularweight, and collecting the material retained by the filter membrane, (c)chromatographing the filter-retained material through Sephadex G-25 andeluting with 0.2M phosphate buffer at pH 7.3, (d) desalting said eluateand chromatographing the desalted product in phosphate buffer at pH 6.3on carboxymethylcellulose and eluting with a NaCl gradient, (e) treatingthe eluate fractions active in the rosette assay with activated charcoaland removing the charcoal, (f) chromatographing the charcoal-treatedmaterial on cellulose and developing first with butanol-pyridinefollowed by 0.01N acetic acid and methanol-chloroform-ammonium hydroxide(20:20:9), and (g) exposing the eluates active in the rosette assay tohigh voltage paper electrophoresis at pH 1.9 and eluting with water. 3.The process of claim 2 wherein step f) is carried out on a celluloseplate with butanol-pyridine (60:30) solvent followed by two-dimensionalchromatography with 0.01N acetic acid in the first dimension andmethanol; chloroform; ammonium hydroxide (20:20:9) in the seconddimension and rechromatographed in methanol; chloroform; ammoniumhydroxide (20:20:9); and wherein step g) is carried out at 40 Volts/cm.and 40 mA in formic acid diluted to pH 1.9.